The overall goal of this research is a detailed characterization of age-dependent changes in the proteome of an oxidation-sensitive area of the brain, the cerebellum. This region controls vestibular function, fine motor behaviors, and some higher cognitive processes such as episodic memory and olfactory perception, all of which are frequently compromised in aged individuals. Furthermore, preliminary studies showed more markers of oxidative stress in aged rat cerebellum than any other brain region. Protein expression will quantitated and post-translational modifications identified and localized in cerebella from F344/BNF1 rats at 5, 22, and 34 months. Analysis will focus specifically on protein phosphorylation and accumulation of protein oxidative modifications such as oxidized thiols, 3-nitrotyrosine, 3, 4-dihydroxyphenylalanine, and 4-hydroxynonenal adducts. Proteomic analysis will be carried out with three cerebellar subcellular fractions involved in neurotransmission and signaling: (i) cerebellar synaptic plasma membranes, (ii) synaptic junctional complexes, and (iii) a multi-subunit glutamate receptor involved in Ca+ and nitric oxide (NO)-dependent cell signaling, the NMDA receptor complex. Analysis of these fractions will yield representative results on composition and potential functional integrity of protein complexes. Yet, selection of these subcellular fractions allows for limited sample sizes (i.e., number of proteins to be analyzed) permitting optimization of quantitative analysis and definitive localization of post-translational modifications through maximum sequence coverage of identified proteins. The Specific Aims are to 1) conduct quantitative proteomic analysis and differential display of age-dependent changes in expression of soluble and membrane proteins in cerebellum, 2) analyze age-dependent phosphorylation status of cerebellar proteins, 3) identify and localize age-dependent oxidative post-translational modifications on cerebellar proteins, and 4) develop novel technologies for enrichment, characterization, and quantitative analysis of DOPA-containing peptides and proteins. Results of these proteomic studies will contribute to emerging databases providing critical insights into brain mechanisms underlying functional decline with age.